The present invention relates to soya (Glycine max) cells and soya cell strains producing isoflavones at a high level and to a process for preparing and isolating isoflavone compounds in high yields. The present invention also pertains to soya plants regenerated from such cell strains and the use thereof for obtaining isoflavone compounds.
Isoflavones are a unique class of plant flavonoids that have a limited distribution in the plant kingdom and are chemically characterized as colorless, crystalline ketones. The most common source for this sort of compounds in nature are soybeans that essentially contain twelve isoflavone isomers: genistein, genistin, 6″-0-malonylgenistin, 6″-0-acetylgenistin, daidzin, daidzein, 6″-0-malonyldaidzein, 6″-0-acetylgenistin; glycitein, glycitin, 6″-0-malonyl-glycitin, 6″-0-acetylglycitin. The principal types of isoflavones found in soybeans are glycones (with sugars) and aglycones (without sugars). Glycones have a glucose molecule attached, and include genistin, daidzin and glycitin, while aglycones are isoflavones without the glucose molecule and include genistein, daidzein and glycitein.
In the recent past isoflavones have been found to exert a number of beneficial effects on living beings upon ingestion. They are deemed to be at least partly responsible for the commonly accepted characteristic of soybeans to reduce the cholesterol level in mammals (Setchell, in McLachlan J A, ed. Estrogens in the Environment II (1985), 69-85). Studies with primates suggest that soya isoflavones may even account for up to about 60-70% of said hypocholesterolemic property (Anthony et al., Circulation 91 (1995), 925). Since coronary heart disease (CHD) is a leading cause of death, especially in the industrialized nations, elevated total cholesterol and low density lipoprotein (LDL) levels are important risk factors for CHD. In humans, soy protein products appear to lower serum total cholesterol levels and low-density lipoprotein (LDL) cholesterol by an average of about 10% when consumed at an average intake level of 47 g soy protein per day.
It has also been suggested that isoflavones participate in the prevention of certain types of cancers. It had statistically been recognized that Japanese women, who due to food intake almost daily consume soy protein, appear to have a quite lower incidence of breast cancer as compared to occidental individuals (Adlercreutz et al., J. Nutr. 125 (1995), 757S-770S). As a scientific confirmation, in animal models, a decrease of mammary tumor formation or inhibition of mammary tumor progression could be revealed when feeding soy protein to rats (Barnes et al., Clin. Biol. Res. 347 (1991), 239-253).
In particular, genistein has been proven to inhibit protein tyrosine kinase (Akiyama et al., J. Biol. Chem. 262 (1987), 5592-5595), to inhibit angiogenesis (Fotsis et al., Proc. Natl. Acad. Sci. USA 90 (1993), 2690-2694), and to induce differentiation in several malignant cell strains (Peterson, J. Nutr. 125 (1995), 784S-789S), all of which are deemed to be important risk factors in cancer development. Genistein and biochanin A are presumed to also inhibit the growth of androgen-dependent and independent prostatic cancer cells (Peterson and Barnes, Prostate 22 (1993), 335-345,) and to act as an antioxidant (Wei et al., Nutr. Cancer 20 (1993), 1-12).
Beyond cancer, it is also thought that at least some of the soya isoflavone fractions are especially beneficial for women in general since they are sources of plant estrogen. It is believed that plant estrogen provides many of the advantages and avoids some of the alleged disadvantages of animal estrogen, so that isoflavones have been proposed to be used for treating menopausal disorders.
So far research has revealed that the pharmacological effects of isoflavone compounds are primarily attributed to their aglycones, such as daidzein or genistein. Specifically, of the soybean isoflavone aglycones genistein has recently been shown to be particularly excellent in physiological activities, including antiosteoporosis activity, antiarteriosclerotic activity, and anticancer activities in the breast, the stomach and the prostate (S. Barnes, Biochem. Biophys. Res. Commun. 179 (1997), 661).
Hence, in view of the known beneficial effects the provision of isoflavones in high amounts is a constant and increasing desideratum.
In the art several methods have been proposed to isolate isoflavones with the first successful attempt dating back to 1931 (Justus Liebigs Ann. Chem 489 (1931), 118).
In U.S. Pat. No. 5,702,752 a process for recovering isoflavone values from a soya molasses feed stream is disclosed. The process comprises subjecting an aqueous soya extract to ultrafiltration to recover the isoflavones as a permeate and further purifying the isoflavones by conventional means.
U.S. Pat. No. 5,821,361 discloses an alternative method for recovering isoflavones from soya molasses. A solution of soya molasses in water is treated at a pH and temperature to convert isoflavone conjugata to isoflavone glucosides, precipitating the isoflavone glucosides and separating the isoflavone glucosides from the solution by filtration and centrifugation.
In addition, JP-05176756A discloses the recovery of isoflavone derivatives from soya based materials by extracting the soya based material with 80% methanol.
A substantial drawback of all the prior art methods resides in that the starting material, the soybean, merely provides a limited amount of isoflavone per unit material utilized to approximately 0.1 to 0.5% dry weight which eventually restricts the amount available and increases the extraction cost. In addition, the level of isoflavones in soybean varies between the years by 3 to 8 fold, which fact is aggravated in that between varieties the level may also vary by about 2 to 3 fold.
A problem of the present invention therefore resides in overcoming the shortcomings of the prior art and providing an improved method for constantly obtaining high amounts of isoflavone compounds. Thus, there is a need for resolving these prior art problems and these are provided by the present invention.